The value of recognizing the toxicity of compounds intended as pharmaceuticals, cosmetics, foods, or other applications where the compounds come into contact with humans or other animals is evident. Because of ethical considerations, not to mention economic ones, of using animal models to predict toxicity against humans, a number of surrogate toxicity tests have been developed permitting more efficient and less controversial approaches to evaluating the capacity of compounds to impact the viability or well being of organisms. For example, a human keratinocyte cell line (SVK-14) has been validated as a model for the effects of mustard vesicants on basal epidermal keratinocytes (Smith, C. N., et al., Human Exp Toxicol (1997) 16:247-253). Normal human epidermal keratinocytes in vitro have also been used to monitor sulfur mustard damage to nuclei and mitochondria (Cook, J. R., et al., Toxicol Pathol (1997) 25:481-486). Apoptosis and necrotic cell death have also been studied as a result of contact with toxic compounds (Kressel, M., et al., Cell Tissue Res (1994) 278:549-556).
The cosmetics industry has been particularly concerned with substituting in vitro tests for in vivo evaluations of toxins. LeClaire, J. et al. Toxicol Lett (1998) 102-103:575-579 describes the experience of L'Oreal in substituting an in vitro system whereby Langerhans cells are introduced into reconstructed epidermis as an alternative test for skin sensitization. The article states that L'Oreal was able totally to ban animal testing on cosmetic products. However, Benassi, L. et al. Contact Dermatitis (1999) 40:38-44 describes a similar attempt to provide a model for whole animal testing. The validity of a monolayer culture system of human keratinocytes was used as a surrogate for in vivo acute skin irritancy tests for several compounds. The results did not entirely correlate, and the authors conclude that this suggests that the keratinocyte monolayer cell culture technique cannot directly replace in vivo methods. Further, Curren, R. et al. in an abstract published in Environ Health Perspect (1998) 106 (Suppl. 2):419-425 reviews the current state of in vitro testing and concludes that in order to progress in the areas of eye and skin irritation, it is necessary to expand knowledge of toxic markers in humans and the biochemical basis of irritation. Thus, attempts to replace in vivo testing with in vitro models have not been entirely successful.
Other approaches will be familiar to the practitioner. Gene expression profiles obtained in response to treating cells or tissues with toxins or other compounds are commonly used as indices of the effects of these compounds. Gene expression can be evaluated either by assessing the pattern of distribution of various mRNAs or by looking directly at the protein levels or activities that result. U.S. Pat. No. 5,811,231 (Xenometrix) discloses a surrogate marker comprising detection of gene transcription from any of several disclosed “stress response” promoters. It is desirable to create surrogates for other kinds of toxicity, such as hypertension, for example, which requires correlating molecular and physiological phenotypes. One such surrogate is disclosed in U.S. Pat. No. 5,569,588 (Acacia) which discloses methods for drug screening by providing a plurality of separately isolated cells, each having an expression system with a different transcriptional regulatory element. Contacting this plurality of cells with a drug candidate and detecting reporter gene product signals from each cell provides a profile of response to the drug with regard to this multiplicity of regulatory elements. In addition, U.S. Pat. No. 5,777,888 (Acacia) describes a system for generating and analyzing a stimulus response output from a collection of signals. The description includes artificial intelligence systems such as expert systems and neural networks for this purpose. These methods, however, rely on gene transcription which is a downstream effect from protein localization as provided hereinbelow.
Since mRNA abundance is now known to be poorly correlated with protein abundance in many instances (Gygi, S. P., et al. Mol Cell Biol (1999) 19:1720-1730 which is incorporated herein by reference), direct examination of the proteins is useful. Standard methods to analyze thousands of proteins in parallel, typically using 2-D gels and mass spectroscopy, are complicated by the frequent occurrence of post-translational modifications. Since the functional impact of many of these modifications is to change subcellular localization, direct visualization of protein location simplifies the analysis. Since degradation can be considered a translocation to the void compartment (typically, via the proteosome degradation machinery), abundance of the protein can be included in the same analysis. Thus, as used here, “intracellular localization” includes a determination of protein levels and changes therein.
Structure activity relationships of compounds having similar toxicities have also been used to predict the behavior of compounds of analogous structures. In addition, cell-based high throughput assays wherein a flow cytometer can be used to identify dead cells, especially after staining with a membrane-impermeable dye, can be used to assess a response. None of these methods is based on assessing intracellular localization.
It has previously been suggested to use multivariate statistical methods to analyze toxicity data obtained from a multiplicity of assays. The National Cancer Institute has assayed over 60,000 compounds for cytotoxicity against 60 cell lines, thus creating an oncology oriented database. Compounds with similar mechanisms of cellular destruction show strong correlations in their activity profiles across the panel. For example, microtubule disrupting agents are particularly effective in some of the cell lines and particularly ineffective on others. A candidate compound which shows a similar pattern has a high probability of exerting an effect on microtubules. This panel was also evaluated with respect to specific responses to the compounds, such as the expression of particular proteins, for example, p53. A cluster of proteins was identified whose expression is correlated with poor efficacy of alkylating agents, but not correlated with efficacy of antitubulin drugs. Thus, by testing a candidate against the 60 cell lines and evaluating the expression of this cluster of proteins, it would be possible to predict whether the compound would or would not be an effective alkylating agent.
Many toxicities encountered clinically affect only a small fraction of the population, but the undetectable effects on the remainder of the population are undoubtedly more widespread. Therefore, assays which assess damage below the clinical threshold and below the threshold detectable by gene transcription are desirable.
There remains a need to develop systems that can serve as accurate surrogates for predicting a wider range of potential toxicity of candidate compounds, assessing the nature of the mechanism by which they exert their toxic effects, and evaluating efficacy of treatment protocols. The present invention offers such a system.